Several mixed-function oxidation systems catalyze inactivation of Escherichia coli glutamine synthetase (GS) and other key metabolic enzymes. The GS inactivation involves the modification of a single histidine residue in each subunit. In the presence of NADPH and molecular oxygen, highly purified preparations of cytochrome P-450 (isozyme 2) and P-450 reductase (both from rabbit liver microsomes) catalyze inactivation of GS. It is proposed that Fe(II) and hydrogen peroxide which are produced by the P-450 system react at the metal binding site on GS to generate an active oxygen species which oxidizes a nearby histidine. In support of this, GS inactivation: (a) is stimulated by FE(III) or CO(II) and is inhibited by EDTA or o-phanthroline, (b) is inhibited by catalase, (c) is stimulated by hexobarbital (which stimulates hydrogen peroxide production), (d) is inhibited by Mn(II) or Zn(II) which also inhibit reduction of Fe(III), (e) is catalyzed by Fe(II) and hydrogen peroxide in absence of the P-450 system, (f) does not require direct interaction of P-450 system with GS because GS inactivation occurs when the P-450 system components and GS are separated by a semipermeable membrane. Furthermore, menadione stimulates GS inactivation by the P-450 system and P-450 itself stimulates GS inactivation by xanthine oxidase. If endogenous catalase is inhibited by azide, rabbit liver microsomes also catalyze GS inactivation. The goal of this project is to provide an understanding of enzyme inactivation by mixed-function oxidation systems and to study its possible biological role in processes such as protein turnover and aging.